Methods and kits for detecting antibodies against an HIV variant

ABSTRACT

The invention concerns a retrovirus extract containing a p25 protein which recognizes immunologically sera of patients afflicted with lymphadenopathy syndrom (LAS) or acquired immune deficiency syndrom (AIDS). It relates to a method and kit for in vivo assay of LAS or AIDS involving contacting sera from patients to be diagnosed for such diseases with said retrovirus extract and by detecting the immunological reaction, if any.

This is a continuation of application Ser. No. 08/424,631, filed Apr.19, 1995, which is a divisional of application Ser. No. 08/019,297,filed Feb. 18, 1993 , which is a division of application Ser. No.07/876,297, filed Apr. 30, 1992, now abandoned, which is a continuationof application Ser. No. 07/117,937, filed Nov. 5, 1987, now U.S. Pat.No. 5,135,864, which is a continuation application of Ser. No.06/785,638, filed Oct. 8, 1995, now U.S. Pat. No. 4,708,818, which is acontinuation application of Ser. No. 06/558,109, filed Dec. 5, 1983, nowabandoned, all of which are incorporated herein by reference.

The invention relates to antigens, means and methods for the diagnosisof lymphadenopathy and acquired immune deficiency syndrome.

BACKGROUND OF THE INVENTION

The acquired immune deficiency syndrome (AIDS) has recently beenrecognized in several countries. The disease has been reported mainly inhomosexual males with multiple partners, and epidemiological studiessuggest horizontal transmission by sexual routes as well as byintravenous drug administration, and blood transfusion. The pronounceddepression of cellular immunity that occurs in patients with AIDS andthe quantitative modifications of subpopulations of their T lymphocytessuggest that T cells or a subset of T cells might be a preferentialtarget for the putative infectious agent. Alternatively, thesemodifications may result from subsequent infections. The depressedcellular immunity may result in serious opportuninistic infections inAIDS patients, many of whom develop Kaposi's sarcoma. However, a pictureof persistent multiple lymphadenopathies has also been described inhomosexual males and infants who may or may not develop AIDS. Thehistological aspect of such lymph nodes is that of reactive hyperplasia.Such cases may correspond to an early or a milder form of the disease.

SUMMARY OF THE INVENTION

It has been found that one of the major etiological agents of AIDS andof lymphadenopathy syndrom (LAS), which is often considered as aprodromic sign of AIDS. should consist of a T-lymphotropic retroviruswhich has been isolated from a lymph node of a homosexual patient withmultiple lymphadenopathies. The virus appears to be distinct from thehuman T-cell leukemia virus (HTLV) family (R. C. Gallo and M. S. Reitz,“J. Natl. Cancer Inst.”, 69 (No. 6), 1209 (1982)). The last mentionedvirus has been known as belonging to the so-called HTLV-1 subgroup.

The patient was a 33-year-old homosexual male who sought medicalconsultation in December 1982 for cervical lymphadenopathy and asthenia(patient 1). Examination showed auxillary and inguinallymphadenopathies. Neither fever nor recent loss of weight were noted.The patient had a history of several episodes of gonorrhea and had beentreated for syphilis in September 1982. During interviews he indicatedthat the had had more than 50 sexual partners per year and had travelledto many countries, including North Africa, Greece, and India. His lasttrip to New York was in 1979.

Laboratory tests indicated positive serology (immunoglobulin G) forcytomegalovirus (CMV) and Epstein-Barr virus. Herpes simplex virus wasdetected in cells from his throat that were cultured on human and monkeycells. A biopsy of a cervical lymph node was performed. One sampleserved for histological examination, which revealed follicularhyperplasia without change of the general structure of the lymph node.Immunohistological studies revealed, in paracortical area, numerous Tlymphocytes (OKT3⁺). Typing of the whole cellular suspension indicatedthat 62 percent of the cells were T lymphocytes (OKT3⁺), 44 percent wereT-helper cells (OKT4⁺)⁻, and 16 percent were suppressor cells (OKTB⁺).

Cells of the same biopsed lymph node were put in culture medium withphytohemagglutinin (PHA), T-cell growth factor (TCGF), and antiserum tohuman α interferon (“The cells were grown in RPMI-1640 mediumsupplemented with antibiotics, 10⁻⁵M β-mercaptoethanol, 10 percent fetalcalf serum, 0.1 percent sheep antibody to human α interferon(neutralizing titer, 7 IU at 10⁻⁵ dilution and 10 percent TCGF, free ofPHA”). The reason for using the antiserum to α-interferon was toneutralize endogenous interferon which is secreted by cells chronicallyinfected by viruses, including retroviruses. In the mouse system, it hadpreviously been shown that anti-serum to interferon could increaseretrovirus production by a factor of 10 to 50 (F. Barré-Sinoussi et al.,“Ann. Microbiol. (Institut Pasteur)” 130B, 349 (1979). After 3 days, theculture was continued in the same medium without PHA. Samples wereregularly taken for reverse transcriptase assay and for examination inthe electron microscope.

After 15 days of culture, a reverse transcriptase activity was detectedin the culture supernatant by using the lionic conditions described forHTLV-I (B. J. Poiesz et al. “Proc. Natl. Acad. Sci. U.S.A.” 77, 7415(1980)). Virus production continued for 15 days and decreasedthereafter, in parallel with the decline of lymphocyte proliferation.Peripheral blood lymphocytes cultured on the same way were consistentlynegative for reverse transcriptase activity, even after 6 weeks.Cytomegalovirus could be detected, upon prolonged co-cultivation withMRC5 cells, in the original biopsy tissue, but not in the cultured Tlymphocytes at any time of the culture.

BRIEF DESCRIPTION OF THE DRAWING

This invention will be more fully described with reference to theFIGURE, which shows curves representative of reverse transcriptaseactivity and tritium-containing uridine activity as a function ofsuccessive fractions of LAV virus in a sucrose gradient.

DETAILED DESCRIPTION

The invention relates to the newly isolated virus as a source of theabove said antigen which will be defined later.

The newly isolated virus, which will hereafter be termed as LAV₁, willhowever be described first.

The virus is transmissible to cultures of T lymphocytes obtained fromhealthy donors. Particularly virus transmission was attempted with theuse of a culture of T lymphocytes established from an adult healthydonor of the Blood Transfusion Center at the Pasteur Institute. On day3, half of the culture was co-cultivate with lymphocytes from the biopsyafter centrifugation of the mixed cell suspensions. Reversetranscriptase activity could be detected in the supernatant on day 15 ofthe coculture but was not detectable on days 5 and 10. The reversetranscriptase had the same characteristics as that released by thepatient's cells and the amount released remained stable for 15 to 20days. Cells of the uninfected culture of the donor lymphocytes did notrelease reverse transcriptase activity during this period or up to 6weeks when the culture was discontinued.

The cell-free supernatant of the infected coculture was used to infect3-day-old cultures of T lymphocytes from two umbilical cords, LC1 andLC5, in the presence of Polybrene (2 μg/ml). After a lag period of 7days, a relatively high titer of reverse transcriptase activity wasdetected in the supernatant of both cord lymphocyte cultures. Identicalcultured, which had not been infected, remained negative. These twosuccessive infections clearly show that the virus could be propagated onnormal lymphocytes from either new-borns or adults.

In the above co-cultures one used either the cells of patient 1 as such(they declined and no longer grew) or cells which had been pre-X-rayedor mitomycin C-treated.

The LAV₁ virus, or LAV₁ virus suspensions, which can be obtained frominfected cultures of lymphocytes have characteristics which distinguishthem completely from other HTLV. These characteristics will be referredto hereafter and, when appropriate, in relation to the drawing. It showscurves representative of variation of reverse transcriptase activity and[³H]uridine activity respectively versus successive fractions of theLAV₁ virus in the sucrose gradient, after ultracentrifugation therein ofthe virus contents of a cell-free supernatant obtained from a culture ofinfected lymphocytes.

The analysis of LAV₁ virus by resorting to reverse transcriptaseactivity can be carried out according to the procedure which was used inrelation to virus from patient 1, on FIG. 1. The results of the analysisare illustrated on FIG. 1. Cord blood T lymphocytes infected with virusfrom patient 1 were labelled for 18 hours with [³H]uridine (28 Ci/mmol,Amersham; 20 μCi/ml). Cell-free supernatant was ultracentrifuged for 1hour at 50,000 rev/min. The pellet was resuspended in 200 μl of NTEbuffer (10 mM tris, pH 7.4, 100 mM NaCl, and 1 mM EDTA) and wascentrifuged over a 3-ml linear sucrose gradient (10 to 60 percent) at55,000 rev/min for 90 minutes in an IEC type SB 498 rotor. Fractions(200 μl) were collected, and 30 μl samples of each fraction were assayedfor DNA RNA dependant polymerase activity with 5 mM Mg²⁺ andpoly(A)-oligo-(dT)₁₂₋₁₈ as template primer; a 20-μl portion of eachfraction was precipitated with 10 percent trichloroacetic acid and thenfiltered on a 0.45-μm Millipore filter. The ³H-labelled acidprecipitable material was measured in a Packard β counter.

That the new virus isolate was a retrovirus was further indicated by itsdensity in the above sucrose gradient, which was 1.16, and by itslabelling with [³H]uridine (FIG. 1). A fast sedimenting RNA appears tobe associated with the LAV₁ virus.

Virus-infected cells from the original biopsy as well as infectedlymphocytes from the first and second viral passages were used todetermine the optimal requirements for reverse transcriptase activityand the template specificity of the enzyme. The results were the same inall instances. The reverse transcriptase activity displayed a strongaffinity for poly(adenylate-oligodeoxythymidylate)[poly(A)-oligo(dT)₁₂₋₁₈], and required Mg²⁺ with an optimalconcentration (5 mM) and an optimal pH of 7.8. The reaction was notinhibited by actinomycin D. This character, as well as the preferentialspecificity for ribosendenylate-deoxythymidylate overdeoxyadenylatedeoxythymidylate, distinguish the viral enzyme fromDNA-dependent polymerases.

Electron microscopy of ultrathin sections of virus-producing cells showstwo types of particles, presumably corresponding to the immature andmature forms of the virus: immature particles are budding at the cellsurface, with a dense crescent in close contact with the plasmamembrane. Occasionally, some particles remain in this state, while beingfreed from the cell surface.

Mature particles have a quite different morphology with a small, dense,eccentric core (mean diameter: 41 nM). Most of virions are round (meandiameter: 139 nM) or ovoid, but in some pictures, especially in theparticles seen in the original culture from which the virus wasisolated, a tailed morphology can also be observed. The latter form canalso be observed in cytoplasmic vesicles which were released in themedium. Such particles are also formed by budding from vesiclemembranes.

Morphology of mature particles is clearly distinct from HTLV, whoselarge core has a mean diameter of 92 nM.

Helper T-lymphocytes (Leu3 cells) form the main target of the virus. Inother words the LAV₁ virus has particular tropism for these cells. Leu 3cells are recognizable by the monoclonal antibodies commercialized byORTHO under the trademark OKT4. In contrast enriched cultures of Leu 2cells, which are mainly suppressor or cytotoxic cells and which arerecognized by the monoclonal antibodies commercialized by ORTHO underthe trademark OKT8 did not produce, when infected under the sameconditions, any detectable RT activity even 6 weeks after virusinfection.

In most cases of AIDS, the ratio of OKT4⁺ over OKT8⁺ cells which isnormally over 1, is depressed to values as low of 0.1 or less.

The LAV₁ virus is also immunologically distinct from previously knownHTLV-1 isolates from cultured T lymphocytes of patients with T lymphomasand T leukemias. The antibodies used were specific for the p19 and p24core proteins of HTLV-1. A monoclonal antibody of p19 (M. Robert-Guroffet al. “J. Exp. Med.” 154, 1957 (1981)) and a polyclonal goat antibodyto p24 (V. S. Kalyanaraman et al. “J. Virol.”, 328, 906 (1981)) wereused in an indirect fluorescence assay against infected cells from thebiopsy of patient 1 and lymphocytes obtained from a healthy donor andinfected with the same virus. The LAV₁ virus-producing cells did notreact with either type of antibody, whereas two lines of cordlymphocytes chronically infected with HTVL 1 (M. Popovic, P. S. Sarin,M. Robert-Guroff, V. S. Kalyanaraman, D. Mann, J. Minowada, R. C. Gallo,“Science” 219, 856 (1983)) and used as controls showed strong surfacefluorescence.

In order to determine which viral antigen was recognized by antibodiespresent in the patient's sera, several immunoprecipitation experimentswere carried out. Cord lymphocytes infected with virus from patient 1and uninfected controls were labelled with [³⁵S]methionine for 20 hours.Cells were lysed with detergents, and a cytoplasmic S10 extract wasmade. Labelled virus released in the supernatant was banded in a sucrosegradient. Both materials were immunoprecipitated by antiserum to HTVL-1′p24, by serum from patient 1, and by serum samples from healthy donors.Immunocomplexes were analyzed by polyacrylamide gel electrophoresisunder cenaturing conditions. A p25 protein present in the virus-infectedcells from patient 1 and in LC1 cells infected with this virus, wasspecifically recognized by serum from patient 1 but not by antiserum toHTLV-1 p24 obtained under similar conditions or serum of normal donors.Conversely the p24 present in control HTLV-infected cell extracts wasrecognized by antibodies to HTLV but not by serum from patient 1.

The main protein (p25) detected after purification of³⁵S-methionine-labelled virus has a molecular weight of about 25,000 (or25K). This is the only protein recognized by the serum of patient 1. Byanalogy with other retroviruses, this major protein was considered to belocated in the viral core.

This can be confirmed in immuno-electron microscopy experiments, whichshow that the patient's serum can agglutinate the viral cores.Conversely, an antiserum raised in rabbit against an ether treated virusdid not precipitate the p25 protein.

The viral origin of other proteins seen in polyacrylamide gelelectrophoresis of purified virus is more difficult to assess. A p15protein could be seen after silver staining, but was much weaker after³⁵S-methionine perhaps due to the paucity of this amino-acid in theprotein. In the higher MW range, a contamination of the virus bycellular proteins, either inside or outside the viral envelope, islikely. A 36K and a 42K protein and a 80K protein were constantly foundto be associated with the purified virus and may represent the majorenvelope proteins.

Ndp19 (or having a molecular weight of about 19,000 was isolated fromLAV₁ extracts.

The invention concerns more particularly the extracts of said virus assoon as they can be recognized immunologically by sera of patientsafflicted with LAS or AIDS. Needless say any type of immunological assaymay be brought into play. By way of example immunofluorescence orimmunoenzymatic assays or radio-immunoprecipitation tests areparticularly suitable.

As a matter of fact and except under exceptional circumstances, sera ofdiseased patients do not recognize the intact LAV₁ virus, or viruseshaving similar phenotypical or immunological properties. The envelopeproteins of the virus appeared as not detectable immunologically by thepatients' sera. However as soon as the core proteins become exposed tosaid sera, the immunological detection becomes possible. Therefore theinvention concerns all extracts of the virus, whether it be the crudestones—particularly mere virus lyzates —or the more purified ones,particularly extracts enriched in the p25 protein or even the purifiedp25 protein or in protein immunologically related therewith. Anypurification procedure may be resorted to. By way of example only, onemay use purification procedures such as disclosed by B. C. Montelero etal, J. of Virology, June 1982, pp. 1029-1038.

The invention concerns more generally extracts of any virus havingsimilar phenotype and immunologically related to that obtained fromLAV₁. Sources of viruses of the LAV type consist of T-lymphocytecultures isolatable from LAS- and AIDS—patients or from haemophiliacs.

In that respect other preferred extracts are those obtained from tworetroviruses obtained by propagation on normal lymphocytes of theretroviruses isolated from:

1) lymph node lymphocytes of a caucasian homosexual with multiplepartners, having extensive Kaposi sarcoma lesions and severe lymphopeniawith practically no OKT4⁺ lymphocytes in his blood;

2) blood lymphocytes of a young B haemophillac presentingneurotoxoplasmosis and OKT4⁺/OKT8⁺ ratio of 0.1.

These two retroviruses have been named IDAV1 and IDAV2 respectively (forImmune Deficiency Associated Virus). Results of partial characterizationobtained so far indicate similarity—if not identity—of IDAV1 and IDAV2to LAV1:

same ionic requirements and template specificities of reversetranscriptase.

same morphology in ultrathin sections.

antigenically related p25 proteins: serum of LAV1 patientimmunoprecipitates p25 from IDAV1 and IDAV2; conversely, serum fromIDAV2 patient immunoprecipitates LAV1 p25.

IDAV1 patient serum seemed to have a lower antibodies titer and gave aweak precipitation band for LAV1 and IDAV1 p25 proteins. The p25 proteinof IDAV1 and IDAV2 was not recognized by HTLV p24 antiserum.

These similarities suggest that all these three isolates belong to thesame group of viruses.

The invention further relates to a method of in vitro diagnosis of LASor AIDS, which comprises contacting a serum or other biological mediumfrom a patient to be diagnosed . . . with a virus extract as abovedefined and detecting the immunological reaction.

Preferred methods brig into play immunoenzymatic or immunofluorescentassays, particularly according to the ELISA technique. Assays may beeither direct or indirect immunoenzymatic or immunofluorescent assays.

Thus the invention also relates to labelled virus extracts whatever thetype of labelling: enzymatic, fluorescent, radioactive, etc..

Such assays include for instance:

depositing determined amounts of the extract according to the inventionin the wells of titration microplate;

introducing in said wells increasing dilutions of the serum to bediagnosed;

incubating the microplate;

washing the microplate extensively;

introducing in the wells of the microplate labelled antibodies directedagainst blood immunoglobulines, and labelling being by an enzymeselected among those which are capable of hydrolysing a substrate,whereby the latter then undergoes a modification of its absorption ofradiations, at least in a determined wavelength band and

detecting, preferably in a comparative manner with respect to a control,the amount of substrate hydrolysis as a measure of the protential risksor effective presence of the disease.

The invention also relates to kits for the abovesaid diagnosis whichcomprise:

an extract or more purified fraction of the abovesaid types of viruses,said extract or fraction being labelled, such as by a radioactive,enzymatic or immunofluorescent label;

human anti-immunoglobulins or protein A (advantageously fixed on awater-insoluble support such as agarose beads);

a lymphocyte extract obtained from a healthy person;

buffers and, if appropriate, substrates for the vizualization of thelabel.

Other features of the invention will further appear as the descriptionproceeds of preferred isolation and culturing procedures of the relevantvirus, of preferred extraction methods of an extract suitable asdiagnostic means, of a preferred diagnosis technique and of the resultsthat can be achieved.

1. VIRUS PROPAGATION

Cultured T-lymphocytes from either umbilical cord or blood, or also bonemarrow cells from healthy, virus negative, adult donors are suitable forvirus propagation.

There is however some variation from individual to individual in thecapacity of lymphocytes to grow the virus. Therefore, it is preferableto select an adult healthy donor, which had no antibodies against thevirus and whose lymphocytes repeatly did not release spontaneouslyvirus, as detected by reverse transcriptase activity (RT) nor expressedviral proteins.

Lymphocytes of the donor were obtained and separated by cytophoresis andstored frozen at −180° C. in liquid nitrogen, in RPMI 1640 medium,supplemented with 50% decomplemented human serum and 10% DMSO untilused.

For viral infection, lymphocytes were put in culture (RPMI 1640 medium)with phytohaemogglutinin (PHA) at the concentration of 5×10⁶ cells/mlfor 3 days.

Then, the medium was removed and cells resuspended in viral suspension(crude supernatant of virus-producing lymphocytes, stored at −80° C.).Optimal conditions of cell/virus concentrations were 2×10⁶ cells for 5to 10,000 cpm of RT activity, the latter determined as previouslydescribed. After 24 hours, cells were centrifuged to remove theunadsorbed virus and resuspended in culture PHA-free medium andsupplemented with PHA-free TCGF (Interleukin 2): (0.5-1 U/ml, finalconcentrations), POLYBREN (Sigma) 2 μg/ml and anti-interferon a sheepserum, inactivated at 56° C. for 30 minutes (0.1% of a serum which isable to neutralize 7U of a leucocyte interferon at a {fraction(1/100,000)} dilution).

Virus production was tested every 3 days by RT activity determination on1 ml samples.

The presence of anti-interferon serum is important in virus production:when lymphocytes were infected in the absence of anti-human-α-interferonserum, virus production, as assayed by RT activity, was very low ordelayed. Since the sheep antiserum used was raised against partlypurified a leucocyte interferon, made according to the Cantelltechnique, the role of components other than interferon cannot beexcluded.

Virus production starts usually from day 9 to 15 after infection, andlasts for 10-15 days. In no cases, the emergence of a continuouspermanent line was observed.

2. VIRUS PURIFICATION

For its use in ELISA, the virus was concentrated by 10%Polyethylenglycol (PEG 6000) precipitation and banded twice toequilibrium in a 20-60% sucrose gradient. The viral band at density 1.16is then recovered and usable as such for ELISA assays.

For use in RIPA radio-immune precipitation assay, purification inisotonic gradients of Metrizamide (sold under the trademark HYCODENZ byNyegaard, Oslo) were found to be preferable. Viral density in suchgradients was very low (1:10-1.11).

Metabolic labelling with ³⁵S-methionine of cells and virus (RIPA)followed by polyacrylamide gel electrophoresis were performed as abovedescribed, except the following modifications for RIPA: virus purifiedin HYCODENZ who lysed in 4 volumes of RIPA containing 500 U/ml ofaprotinin. Incubation with 5 μl of serum to be tested was made for 1hour at 37° C. and then 18 hours at +4° C. Further incubation of theimmunocomplexes with protein A SEPHAROSE beads was for 3 hours at +4° C.

3. PREPARATION OF THE VIRUS EXTRACT FOR ELISA ASSAYS

Virus purified in sucrose gradient as above described, is lysed in RIPAbuffer (0.5% SDS) and coated on wells of microtest plates (Nunc).

Preferred conditions for the ELISA assay are summed up hereafter.

After addition to duplicate wells of serial dilutions of each serum tobe tested, the specifically fixed IgGs are revealed by goat anti-humanIgG coupled with peroxydase. The enzymatic reaction is carried out onortho-phenylene-diamine as substrate and read with an automaticspectrophotometer at 492 nM.

On the same plate each serum is tested on a control antigen (a crudecytoplasmic lysate of uninfected T-lymphocytes from the same donor) isused in order to eliminate unspecific binding, which can be high withsome sera.

Sera are considered as positive (antibodies against the virus) when thedifference between O.D. against the viral antigen and O.D. againstcontrol cellular antigen was at least 0.30.

Hereafter there is disclosed a specific test for assaying the abovementioned disease or exposure to disease risks.

Method

This ELISA test is for detecting and titration of seric anti-retrovirustype LAV antibodies.

It comprises carrying out a competition test between a viral antigen(cultivated on T lymphocytes) and a control antigen constituted by alysate of the same through non-infected lymphocytes.

The binding of the antibodies on the two antigens is revealed by the useof a human antiglobulin labelled with an enzyme which itself is revealedby the addition of a corresponding substrate.

Preparation of the Viral Antigen

The cellular cultures which are used are T lymphocytes of human originwhich come from:

umbilical cord blood,

bone marrow,

blood of a healthy donor.

After infection of the cells by the virus, the supernatant of theinfected cell culture is used. It is concentrated by precipitating with10% PEG, then purified (two or three times) on a (20-60%) sucrosegradient by ultracentrifugation to equilibrium.

The viral fractions are gathered and concentrated by centrifugation at50 000 rotations per minute for 60 minutes.

The sedimented virus is taken in a minimum volume of buffer HTE at pH7.4 (Tris 0.01M, NaCl 0.1M, EDTA 0.001M).

The proteic concentration is determined by the Lowry method.

The virus is then lysed by a (RIPA+SDS) buffer (0.5% final) for 15minutes at 37° C.

Preparation of the Control Antigen

The non-infected lymphocytes are cultured according to the preceedingconditions for from 5 to 10 days. They are centrifuged at low speed andlysed in the RIPA buffer in the presence of 5% of the productcommercialized under the name of ZYMOFREN (Special (500 μ/ml). After astay of 15 minutes at 4° C. with frequent stirrings with vortex, thelysate is centrifuged at 10 000 rotations per minute. The supernatantconstitutes the control antigen. Its concentration in protein ismeasured by the Lowry method.

Reagents

1—Plates=NUNC−special controlled ELISA

2—Buffer PBS: pH 7.5

3—TWEEN 20

4—Carbonate buffer: pH=9.6 (CO₃Ha₂=0.2M (CO₃HN_(a)=0.2M

5—Non foetal calf serum: which is stored in frozen state (BIOPRO).

6—Bovine serum albumine (BSA) SIGMA (fraction V)

7—Human anti IgG (H+L) labelled with peroxydase PASTEUR, in tubes of 1ml preserved at 4° C.

8—Washing buffer=PBS buffer, pH 7.5+0.05% TWEEN 20

Dilution of the conjugate is carried out at the dilution indicated inPBS buffer+TWEEN 20 (0.05%)+bovine albumine 0.5 g per 100 ml

9—Dilution buffer of sera=PBS buffer+0.05% TWEEN 20−0.5 g BSA bovineserum albumine per 100 ml

10—Substrate=OPD

Citrate buffer pH=5.6 trisodic citrate (C₆H₅Na₄O₃, 2H₂O), 0.05M; citricacid (C₆H₈O₇, 1H₂O), 0.05M.

Hydrogen peroxyde=at 30% (110 volumes)−used at 0.03% when using citratebuffer.

Orthophenylene diamine=SIGMA

75 mg per 25 ml of buffer—which is diluted in buffer extemporaneously.

Preparation of the Plates

The plates which are used have 96U-shaped wells (NUNC=ELISA). Theyinclude 12 rows of 8 wells each, numbered from 1 to 12.

The distribution of antigens is as follows:

100 μl of the viral antigen, diluted in carbonate buffer at pH 9.6, aredeposited in each of the wells of rows (marked Θ)

1-2-5-6-9-10

100 μl of the control antigen, diluted in carbonate buffer at pH 9.6,are deposited in each of the wells of rows (marked Θ)

3-4-7-8-11-12.

The dilution of the viral antigen is titrated at each viral production.Several dilutions of viral antigen are tested and compared to positiveand negative known controls (at several dilutions) and to human anti-IgGlabelled with peroxydase, the latter being also tested at severaldilutions.

As a rule, the proteic concentration of the preparation is of 5 to 2.5μg/ml.

The same proteic concentration is used for the control antigen.

The plates are closed with a plastic lid and are incubated overnight at4° C.

Then they are put once in distilled water and centrifuged. The wells arethen filled with 300 μl of non foetal calf serum at 20% in PBS buffer.

The incubation lasts 2 hours at 37° C. (covered plates).

The plates are washed 3 times in PBS buffer with TWEEN 20, 0.05% (PBS-twbuffer):

first washing 300 μl

second and third washing 200 μl/well.

The plates are carefully dried and sealed with an adhesive plastic film.They can be stored at −80° C.

ELISA Reaction: Antibody Titer Assay

After defreezing, the plates are washed 3 times in PBS-TWEEN. They arecarefully dried.

The positive and negative control sera as well as the tested sera arediluted first in the tube, with PBS-TWEEN containing 0.5% bovinealbumine.

The chosen dilution is {fraction (1/40)}.

100 μl of each serum are deposited in duplicate on the viral antigen andin duplicate on the control antigen.

the same is carried out for the positive and negative diluted sera.

100 μl of PBS+TWEEN+bovine serum albumine are introduced in two wells Θand in two wells Θ to form the conjugated controls.

The plates equipped with their lids are incubated for 1.5 h at 37° C.

They are washed 4 times in PBS+TWEEN 0.05%. −100 μl of human anti-IgG(labelled with peroxydase) at the chosen dilution are deposited in eachwell and incubated at 37° C.

The plates are again washed 5 times with the (PBS+TWEEN) buffer. Theyare carefully dried.

Revealing the enzymatic reaction is carried out by means of aorthophenylene-diamine substrate (0.05% in citrate buffer pH 5.6containing 0.03% of H₂O₂).

100 μl of substrate are distributed in each well.

The plates are left in a dark room 20 minutes at the laboratorytemperature.

Reading is carried out on a spectrophotometer (for microplates) at 492nm.

Sera deemed as containing antibodies against the virus are those whichgive a ODD (optical density difference=optical density of viral antigenless optical density of control antigen) equal or higher to 0.30.

This technique enables a qualitative titration as well as a quantitativeone. For this purpose, it is possible either to use several dilutions ofthe serum to be assayed, or to compare a dilution of the serum with arange of controls tested under the same conditions.

The table hereafter provides first results of serological investigationsfor LAV antibodies, carried out by using the above exemplified ELISAassay.

FIRST RESULTS OF SEROLOGICAL INVESTIGATIONS FOR LAV ANTIBODIES IN FRANCEELISA-HTLV1** Total ELISA-LAV (Biotech) examined positive % positivepositive % positive Lymphadeno- 35 22  (63)   5*** (14) pathy patients*Healthy 40 7 (17) 1  (3) homosexuals Control 54 1 (1,9) 0 (<2,6)population *28 homosexuals 3 Haitians (1 woman) 4 toxicomans (2 women)**The number of positive sera is probably overestimated in this test,since no control of unspecific binding could be done. ***Out of the 5LAS HTLV1 positive, 3 were born in Haiti, 1 had stayed for a long timein Haiti and 1 had made several travels to USA. All of them had alsoantibodies against LAV.

The table shows clearly high prevalence of LAV antibodies in thehomosexual patients with LAS, the very low incidence in the normalpopulation and also a moderate spread of virus infection in stillhealthy homosexuals. In the latter group, all the positive individualshad a high number of partners (>50 per year). The incidence of HTLVantibodies was very low in all three groups (determined by using acommercial ELISA test (Biotech)). The groups of AIDS patients gave lessinterpretable results: approximatively 20% had LAV antibodies, but someof the sera were taken at a very late stage of the disease, with apossible negativation of the humoral response.

It should further be mentioned that lymphocytes of all LAS patients donot produce detectable amounts of LAV-type virus. Particularly cells oflymph nodes from 6 more LAS patients were put in culture and tested forvirus production, as described for patient 1. No virus release could bedetected by RT activity. However, a p25 protein recognized by the serumof the first patient could be detected in cytoplasmic extracts of theT-cells labelled with ³⁵S-methionine in 3 other cases. This suggestspartial expression of a similar virus in such cases. Moreover, all (6/6)of these patients had antibodies against LAV p25 proteins, indicatingthat they all had been infected with a similar or identical virus.

Interestingly, in lymphocytes of one of the patients (patient 2), therewas a weak but definite immunoprecipitation of a band of similar size(p24-p25) with goat antiserum raised against HTLV1. Similarly, thepatient's serum had antibodies against both HTLV and LAV, suggesting adouble infection by both viruses. Such cases seem rather infrequent.

The invention finally also relates to the biological reagents that canbe formed by the LAV extracts containing the p25 protein or by thepurified p25 protein, particularly for the production of antibodiesdirected against p25 in animals or of monoclonal antibodies. Theseantibodies are liable of forming useful tools in the further study ofantigenic determinants of LAV viruses or LAV-related viruses.

It is acknowledged that the OKT designations which have been used withrespect to the designation of some sub-sets of lymphocytes or relatedmonoclonal antibodies by way of ease of language, should in no way beopposed to the validity of any corresponding trademark, whetherregistered or not by its owner.

It should further be mentioned that the viral extracts, particularlyviral lysates or enriched fractions can also be defined by reference totheir immunological relationship or similitude with the extracts orenriched fractions containing a p25 protein as obtainable from thestrain LAV1, IDAV1 or IDAV2. Thus any protein fraction which is capableof giving similar patterns of immunological reaction as do the proteinextracts of LAV1, IDAV1 or IDAV2 with the same sera, must be consideredas equivalent thereof and, accordingly, be deemed as encompassed by thescope of the claims which follow. A similar conclusion extends of courseto the diagnostic means (process and kits) which may make use of suchequivalent protein extracts.

The LAV1 virus has been deposited at the “Collection Nationale desCultures de Micro-organismes” (C.N.C.M.) under n° I-232 on Jul. 14, 1983and IDAV1 and IDAV 2 viruses have been deposited at the C.N.C.M.,Institut Pasteur, 28 rue de Docteur Roux, 75724 Paris Cedex 15, France,on Sep. 15, 1983 under n° I-240 and I-241, respectively. The inventionencompasses as well the extracts of mutants of variants of the abovedeposited strains as long as they possess substantially the sameimmunological properties.

We claim:
 1. A method for the in vitro identification of humanimmunodeficiency virus type 1 (HIV-1) comprising: subjecting cultures ofinfected and uninfected human lymphocytes to a protein labelingreaction; lysing said labeled cultures of lymphocytes; contacting saidlysed lymphocytes with patient serum comprising an antibody that bindsto p25 of HIV-1 viruses to form immunocomplexes; separating saidimmunocomplexes; and detecting labeled proteins in said separatedimmunocomplexes, wherein the detection of labeled HIV-1 proteins in saidinfected culture is indicative of the presence of a cell infected byHIV-1 in said culture.
 2. The method of claim 1, wherein saidimmunocomplexes are separated by precipitation with protein A.
 3. Themethod of claim 1, wherein said labeled proteins are resolved on aplyacrylamide gel under denaturing conditions.
 4. The method of claim 1,wherein said proteins are radiolabeled.
 5. The method of claim 4,wherein said proteins are radiolabeled with ³⁵S-methionine.
 6. A methodfor the in vitro identification of a human immunodeficiency virus type 1(HIV-1) comprising: contacting lysed human lymphocytes infected withHIV-1 and uninfected human lymphocytes with patient serum comprising anantibody that binds to p25 of HIV-1 viruses to form immunocomplexes;contacting said immunocomplexes with a fluorescently-labeled antibodythat binds to said immunocomplexes; and determining the presence ofimmunocomplexes by detecting fluorescence in the infected and uninfectedsamples, wherein the presence of immunocomplexes is indicative of thepresence of human lymphocytes infected with HIV-1.
 7. A method for thein vitro identification of antibodies that bind to humanimmunodeficiency virus type 1 (HIV-1) in patient serum comprising:contacting lysed human lymphocytes infected with HIV-1 and uninfectedhuman lymphocytes with patient serum comprising an antibody that bindsto p25 of HIV-1 viruses to form immunocomplexes; contacting saidimmunocomplexes with a fluorescently-labeled antibody that binds to saidimmunocomplexes; and determining the presence of immunocomplexes bydetecting fluorescence in the infected and uninfected samples, whereinthe presence of immunocomplexes is indicative of the presence ofantibodies that bind to human immunodeficiency virus type 1 (HIV-1) insaid patient serum.
 8. A method for the in vitro identification of ahuman immunodeficiency virus type 1 (HIV-1) comprising: contacting lysedhuman lymphocytes infected with HIV-1 and uninfected human lymphocyteswith patient serum comprising an antibody that binds to p25 of HIV-1viruses to form immunocomplexes, wherein said antibody is fluorescentlylabeled; and determining the presence of immunocomplexes by detectingfluorescence in the infected and uninfected samples, wherein thepresence of immunocomplexes is indicative of the presence of humanlymphocytes infected with HIV-1.
 9. A method for the in vitroidentification of a human immunodeficiency virus type 1 (HIV-1)comprising: contacting lysed human lymphocytes infected with HIV-1 anduninfected human lymphocytes with patient serum comprising an antibodythat binds to p25 of HIV-1 viruses to form immunocomplexes; contactingsaid immunocomplexes with an enzymatically-labeled antibody that bindsto said immunocomplexes; and determining the presence of immnocomplexesby an enzymatic reaction in the infected and uninfected samples, whereinthe presence of immunocomplexes is indicative of the presence of humanlymphocytes infected with HIV-1.
 10. A method for the in vitroidentification of antibodies that bind to human immunodeficiency virustype 1 (HIV-1) in patient serum comprising: contacting lysed humanlymphocytes infected with HIV-1 and uninfected human lymphocytes withpatient serum comprising an antibody that binds to p25 of HIV-1 virusesto form immunocomplexes; contacting said immunocomplexes with anenzymatically-labeled antibody that binds to said immunocomplexes; anddetermining the presence of immunocomplexes by an enzymatic reaction inthe infected and uninfected samples, wherein the presence ofimmunocomplexes is indicative of the presence of antibodies that bind tohuman immunodeficiency virus type 1 (HIV-1) in said patient serum.
 11. Amethod for the in vitro identification of a human immunodeficiency virustype 1 (HIV-1) comprising: contacting lysed human lymphocytes infectedwith HIV-1 and uninfected human lymphocytes with patient serumcomprising an antibody that binds to p25 of HIV-1 viruses to formimmunocomplexes. wherein said antibody is enzymatically labeled; anddetermining the presence of immunocomplexes by an enzymatic reaction inthe infected and uninfected samples, wherein the presence ofimmunocomplexes is indicative of the presence of human lymphocytesinfected with HIV-1.
 12. A method for the in vitro identification of ahuman immunodeficiency virus type 1 (HIV-1) comprising: contacting lysedhuman lymphocytes infected with HIV-1 and uninfected human lymphocyteswith patient serum comprising an antibody that binds to p25 of HIV-1viruses to form immunocomplexes; contacting said immunocomplexes with alabeled antibody that binds to said immunocomplexes; and determining thepresence of labeled immunocomplexes in the infected and uninfectedsamples, wherein the presence of immunocomplexes is indicative of thepresence of human lymphocytes infected with HIV-1.
 13. A method for thein vitro identification of antibodies that bind to humanimmunodeficiency virus type 1 (HIV-1) in patient serum comprising:contacting lysed human lymphocytes infected with HIV-1 and uninfectedhuman lymphocytes with patient serum comprising an antibody that bindsto p25 to HIV-1 viruses to form immunocomplexes; contacting saidimmunocomplexes with a labelled antibody that binds to saidimmunocomplexes; and determining the presence of labeled immunocomplexesin the infected and uninfected samples, wherein the presence ofimmunocomplexes is indicative of the presence of antibodies that bind tohuman immunodeficiency virus type 1 (HIV-1) in said patient serum.
 14. Amethod for the in vitro identification of a human immunodeficiency virustype 1 (HIV-1) comprising: contacting lysed human lymphocytes infectedwith HIV-1 and uninfected human lymphocytes with patient serumcomprising an antibody that binds to p25 of HIV-1 viruses to formimmunocomplexes, wherein said antibody is labeled; and determining thepresence of labeled immunocomplexes in the infected and uninfectedsamples, wherein the presence of immunocomplexes is indicative of thepresence of human lymphocytes infected with HIV-1.